Turn-on RNA Mango Beacons for trans-acting fluorogenic nucleic acid detection.

The Mango I and II RNA aptamers have been widely used in vivo and in vitro as genetically encodable fluorogenic markers that undergo large increases in fluorescence upon binding to their ligand, TO1-Biotin. However, while studying nucleic acid sequences, it is often desirable to have trans-acting probes that induce fluorescence upon binding to a target sequence. Here, we rationally design three types of light-up RNA Mango Beacons based on a minimized Mango core that induces fluorescence upon binding to a target RNA strand. Our first design is bimolecular in nature and uses a DNA inhibition strand to prevent folding of the Mango aptamer core until binding to a target RNA. Our second design is unimolecular in nature, and features hybridization arms flanking the core that inhibit G-quadruplex folding until refolding is triggered by binding to a target RNA strand. Our third design builds upon this structure, and incorporates a self-inhibiting domain into one of the flanking arms that deliberately binds to, and precludes folding of, the aptamer core until a target is bound. This design separates G-quadruplex folding inhibition and RNA target hybridization into separate modules, enabling a more universal unimolecular beacon design. All three Mango Beacons feature high contrasts and low costs when compared to conventional molecular beacons, with excellent potential for in vitro and in vivo applications.

Harmonizing the growing fluorogenic RNA aptamer toolbox for RNA detection and imaging.

The field of fluorogenic RNA aptamers is a burgeoning research area that aims to address the lack of naturally fluorescent RNA molecules for RNA detection and imaging. These small RNA tags bind to their fluorogenic ligands resulting in significant fluorescent enhancement, leading to a molar brightness comparable to or exceeding that of fluorescent proteins. In the past decade, multiple light-up RNA aptamer systems have been isolated that bind to a broad range of ligands involving several distinct mechanisms of fluorogenicity. This review discusses the selection methods used to isolate fluorogenic RNA aptamers. More than seventy fluorogenic aptamer:ligand pairs are evaluated using objective parameters (e.g., molar brightness, binding affinity, fluorophore exchange capabilities and other details). General guidelines for choosing fluorescent RNA tools, with an emphasis on single-molecule detection and multi-colour imaging applications are provided. Lastly the importance of global standards for evaluating fluorogenic RNA aptamer systems is discussed.

E. coli 6S RNA complexed to RNA polymerase maintains product RNA synthesis at low cellular ATP levels by initiation with noncanonical initiator nucleotides.

The E. coli 6S RNA is an RNA polymerase (RNAP) inhibitor that competes with σ70-dependent DNA promoters for binding to RNAP holoenzyme (RNAP:σ70). The 6S RNA when bound is then used as a template to synthesize a short product RNA (pRNA; usually 13-nt-long). This pRNA changes the 6S RNA structure, triggering the 6S RNA:pRNA complex to release and allowing DNA-dependent housekeeping gene expression to resume. In high nutrient conditions, 6S RNA turnover is extremely rapid but becomes very slow in low nutrient environments. This leads to a large accumulation of inhibited RNAP:σ70 in stationary phase. As pRNA initiates synthesis with ATP, we and others have proposed that the 6S RNA release rate strongly depends on ATP levels as a proxy for sensing the cellular metabolic state. By purifying endogenous 6S RNA:pRNA complexes using RNA Mango and using reverse transcriptase to generate pRNA-cDNA chimeras, we demonstrate that 6S RNA:pRNA formation can be simultaneous with 6S RNA 5' maturation. More importantly, we find a dramatic accumulation of capped pRNAs during stationary phase. This indicates that ATP levels in stationary phase are low enough for noncanonical initiator nucleotides (NCINs) such as NAD+ and NADH to initiate pRNA synthesis. In vitro, mutation of the conserved 6S RNA template sequence immediately upstream of the pRNA transcriptional start site can increase or decrease the pRNA capping efficiency, suggesting that evolution has tuned the biological 6S RNA sequence for an optimal capping rate. NCIN-initiated pRNA synthesis may therefore be essential for cell viability in low nutrient conditions.

Single-Molecule RNA Imaging Using Mango II Arrays.

In recent years, fluorogenic RNA aptamers, such as Spinach, Broccoli, Corn, Mango, Coral, and Pepper have gathered traction as an efficient alternative labeling strategy for background-free imaging of cellular RNAs. However, their application has been somewhat limited by relatively inefficient folding and fluorescent stability. With the recent advent of novel RNA-Mango variants which are improved in both fluorescence intensity and folding stability in tandem arrays, it is now possible to image RNAs with single-molecule sensitivity. Here we discuss the protocol for imaging Mango II tagged RNAs in both fixed and live cells.

Processive RNA polymerization and promoter recognition in an RNA World.

Early life is thought to have required the self-replication of RNA by RNA replicases. However, how such replicases evolved and subsequently enabled gene expression remains largely unexplored. We engineered and selected a holopolymerase ribozyme that uses a sigma factor-like specificity primer to first recognize an RNA promoter sequence and then, in a second step, rearrange to a processive elongation form. Using its own sequence, the polymerase can also program itself to polymerize from certain RNA promoters and not others. This selective promoter-based polymerization could allow an RNA replicase ribozyme to define "self" from "nonself," an important development for the avoidance of replicative parasites. Moreover, the clamp-like mechanism of this polymerase could eventually enable strand invasion, a critical requirement for replication in the early evolution of life.

Microchip RT-PCR Detection of Nasopharyngeal SARS-CoV-2 Samples.

Fast, accurate, and reliable diagnostic tests are critical for controlling the spread of the coronavirus disease 2019 (COVID-19) associated with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. The current gold standard for testing is real-time PCR; however, during the current pandemic, supplies of testing kits and reagents have been limited. We report the validation of a rapid (30 minutes), user-friendly, and accurate microchip real-time PCR assay for detection of SARS-CoV-2 from nasopharyngeal swab RNA extracts. Microchips preloaded with COVID-19 primers and probes for the N gene accommodate 1.2-µL reaction volumes, lowering the required reagents by 10-fold compared with tube-based real-time PCR. We validated our assay using contrived reference samples and 21 clinical samples from patients in Canada, determining a limit of detection of 1 copy per reaction. The microchip real-time PCR provides a significantly lower resource alternative to the Centers for Disease Control and Prevention-approved real-time RT-PCR assays with comparable sensitivity, showing 100% positive and negative predictive agreement of clinical samples.

RNA Peach and Mango: Orthogonal two-color fluorogenic aptamers distinguish nearly identical ligands.

Two-channel fluorogenic RNA aptamer-based imaging is currently challenging. While we have previously characterized the Mango series of aptamers that bind tightly and specifically to the green fluorophore TO1-Biotin, the next aim was to identify an effective fluorogenic aptamer partner for two-color imaging. A competitive in vitro selection for TO3-Biotin binding aptamers was performed resulting in the Peach I and II aptamers. Remarkably, given that the TO1-Biotin and TO3-Biotin heterocycles differ by only two bridging carbons, these new aptamers exhibit a marked preference for TO3-Biotin binding relative to the iM3 and Mango III A10U aptamers, which preferentially bind TO1-Biotin. Peach I, like Mango I and II, appears to contain a quadruplex core isolated by a GAA^A type tetraloop-like adaptor from its closing stem. Thermal melts of the Peach aptamers reveal that TO3-Biotin binding is cooperative, while TO1-Biotin binding is not, suggesting a unique and currently uncharacterized mode of ligand differentiation. Using only fluorescent measurements, the concentrations of Peach and Mango aptamers could be reliably determined in vitro. The utility of this orthogonal pair provides a possible route to in vivo two-color RNA imaging.

Fluorogenic aptamers resolve the flexibility of RNA junctions using orientation-dependent FRET.

To further understand the transcriptome, new tools capable of measuring folding, interactions, and localization of RNA are needed. Although Förster resonance energy transfer (FRET) is an angle- and distance-dependent phenomenon, the majority of FRET measurements have been used to report distances, by assuming rotationally averaged donor-acceptor pairs. Angle-dependent FRET measurements have proven challenging for nucleic acids due to the difficulties in incorporating fluorophores rigidly into local substructures in a biocompatible manner. Fluorescence turn-on RNA aptamers are genetically encodable tags that appear to rigidly confine their cognate fluorophores, and thus have the potential to report angular-resolved FRET. Here, we use the fluorescent aptamers Broccoli and Mango-III as donor and acceptor, respectively, to measure the angular dependence of FRET. Joining the two fluorescent aptamers by a helix of variable length allowed systematic rotation of the acceptor fluorophore relative to the donor. FRET oscillated in a sinusoidal manner as a function of helix length, consistent with simulated data generated from models of oriented fluorophores separated by an inflexible helix. Analysis of the orientation dependence of FRET allowed us to demonstrate structural rigidification of the NiCo riboswitch upon transition metal-ion binding. This application of fluorescence turn-on aptamers opens the way to improved structural interpretation of ensemble and single-molecule FRET measurements of RNA.

Structure-Guided Engineering of the Homodimeric Mango-IV Fluorescence Turn-on Aptamer Yields an RNA FRET Pair.

Fluorescent RNA aptamers have been used in cells as biosensor reporters and tags for tracking transcripts. Recently, combined SELEX and microfluidic fluorescence sorting yielded three aptamers that activate fluorescence of TO1-Biotin: Mango-II, Mango-III, and Mango-IV. Of these, Mango-IV was best at imaging RNAs in both fixed and live mammalian cells. To understand how Mango-IV achieves activity in cells, we determined its crystal structure complexed with TO1-Biotin. The structure reveals a domain-swapped homodimer with two independent G-quadruplex fluorophore binding pockets. Structure-based analyses indicate that the Mango-IV core has relaxed fluorophore specificity, and a tendency to reorganize binding pocket residues. These molecular properties may endow it with robustness in the cellular milieu. Based on the domain-swapped structure, heterodimers between Mango-IV and the fluorescent aptamer iSpinach, joined by Watson-Crick base pairing, were constructed. These exhibited FRET between their respective aptamer-activated fluorophores, advancing fluorescent aptamer technology toward multi-color, RNA-based imaging of RNA coexpression and colocalization.

Live cell imaging of single RNA molecules with fluorogenic Mango II arrays.

RNA molecules play vital roles in many cellular processes. Visualising their dynamics in live cells at single-molecule resolution is essential to elucidate their role in RNA metabolism. RNA aptamers, such as Spinach and Mango, have recently emerged as a powerful background-free technology for live-cell RNA imaging due to their fluorogenic properties upon ligand binding. Here, we report a novel array of Mango II aptamers for RNA imaging in live and fixed cells with high contrast and single-molecule sensitivity. Direct comparison of Mango II and MS2-tdMCP-mCherry dual-labelled mRNAs show marked improvements in signal to noise ratio using the fluorogenic Mango aptamers. Using both coding (ß-actin mRNA) and long non-coding (NEAT1) RNAs, we show that the Mango array does not affect cellular localisation. Additionally, we can track single mRNAs for extended time periods, likely due to bleached fluorophore replacement. This property makes the arrays readily compatible with structured illumination super-resolution microscopy.

RNA detection with high specificity and sensitivity using nested fluorogenic Mango NASBA.

There is a pressing need for nucleic acid-based assays that are capable of rapidly and reliably detecting pathogenic organisms. Many of the techniques available for the detection of pathogenic RNA possess one or more limiting factors that make the detection of low-copy RNA challenging. Although RT-PCR is the most commonly used method for detecting pathogen-related RNA, it requires expensive thermocycling equipment and is comparatively slow. Isothermal methods promise procedural simplicity but have traditionally suffered from amplification artifacts that tend to preclude easy identification of target nucleic acids. Recently, the isothermal SHERLOCK system overcame this problem by using CRISPR to distinguish amplified target sequences from artifactual background signal. However, this system comes at the cost of introducing considerable enzymatic complexity and a corresponding increase in total assay time. Therefore, simpler and less expensive strategies are highly desirable. Here, we demonstrate that by nesting NASBA primers and modifying the NASBA inner primers to encode an RNA Mango aptamer sequence we can dramatically increase the sensitivity of NASBA to 1.5 RNA molecules per microliter. As this isothermal nucleic acid detection scheme directly produces a fluorescent reporter, real-time detection is intrinsic to the assay. Nested Mango NASBA is highly specific and, in contrast to existing RNA detection systems, offers a cheap, simple, and specific way to rapidly detect single-molecule amounts of pathogenic RNA.

Fluorescent Visualization of Mango-tagged RNA in Polyacrylamide Gels via a Poststaining Method.

Native and denaturing polyacrylamide gels are routinely used to characterize ribonucleoprotein (RNP) complex mobility and to measure RNA size, respectively. As many gel-imaging techniques use nonspecific stains or expensive fluorophore probes, sensitive, discriminating, and economical gel-imaging methodologies are highly desirable. RNA Mango core sequences are small (19-22 nt) sequence motifs that, when closed by an arbitrary RNA stem, can be simply and inexpensively appended to an RNA of interest. These Mango tags bind with high affinity and specificity to a thiazole-orange fluorophore ligand called TO1-Biotin, which becomes thousands of times more fluorescent upon binding. Here we show that Mango I, II, III, and IV can be used to specifically image RNA in gels with high sensitivity. As little as 62.5 fmol of RNA in native gels and 125 fmol of RNA in denaturing gels can be detected by soaking gels in an imaging buffer containing potassium and 20 nM TO1-Biotin for 30 min. We demonstrate the specificity of the Mango-tagged system by imaging a Mango-tagged 6S bacterial RNA in the context of a complex mixture of total bacterial RNA.

Structure and functional reselection of the Mango-III fluorogenic RNA aptamer.

Several turn-on RNA aptamers that activate small-molecule fluorophores have been selected in vitro. Among these, the ~30 nucleotide Mango-III is notable because it binds the thiazole orange derivative TO1-Biotin with high affinity and fluoresces brightly (quantum yield 0.55). Uniquely among related aptamers, Mango-III exhibits biphasic thermal melting, characteristic of molecules with tertiary structure. We report crystal structures of TO1-Biotin complexes of Mango-III, a structure-guided mutant Mango-III(A10U), and a functionally reselected mutant iMango-III. The structures reveal a globular architecture arising from an unprecedented pseudoknot-like connectivity between a G-quadruplex and an embedded non-canonical duplex. The fluorophore is restrained into a planar conformation by the G-quadruplex, a lone, long-range trans Watson-Crick pair (whose A10U mutation increases quantum yield to 0.66), and a pyrimidine perpendicular to the nucleobase planes of those motifs. The improved iMango-III and Mango-III(A10U) fluoresce ~50% brighter than enhanced green fluorescent protein, making them suitable tags for live cell RNA visualization.

Crystal Structures of the Mango-II RNA Aptamer Reveal Heterogeneous Fluorophore Binding and Guide Engineering of Variants with Improved Selectivity and Brightness.

Several RNA aptamers that bind small molecules and enhance their fluorescence have been successfully used to tag and track RNAs in vivo, but these genetically encodable tags have not yet achieved single-fluorophore resolution. Recently, Mango-II, an RNA that binds TO1-Biotin with ∼1 nM affinity and enhances its fluorescence by >1500-fold, was isolated by fluorescence selection from the pool that yielded the original RNA Mango. We determined the crystal structures of Mango-II in complex with two fluorophores, TO1-Biotin and TO3-Biotin, and found that despite their high affinity, the ligands adopt multiple distinct conformations, indicative of a binding pocket with modest stereoselectivity. Mutational analysis of the binding site led to Mango-II(A22U), which retains high affinity for TO1-Biotin but now discriminates >5-fold against TO3-biotin. Moreover, fluorescence enhancement of TO1-Biotin increases by 18%, while that of TO3-Biotin decreases by 25%. Crystallographic, spectroscopic, and analogue studies show that the A22U mutation improves conformational homogeneity and shape complementarity of the fluorophore-RNA interface. Our work demonstrates that even after extensive functional selection, aptamer RNAs can be further improved through structure-guided engineering.

High-throughput detection of RNA processing in bacteria.

BACKGROUND: Understanding the RNA processing of an organism's transcriptome is an essential but challenging step in understanding its biology. Here we investigate with unprecedented detail the transcriptome of Pseudomonas aeruginosa PAO1, a medically important and innately multi-drug resistant bacterium. We systematically mapped RNA cleavage and dephosphorylation sites that result in 5'-monophosphate terminated RNA (pRNA) using monophosphate RNA-Seq (pRNA-Seq). Transcriptional start sites (TSS) were also mapped using differential RNA-Seq (dRNA-Seq) and both datasets were compared to conventional RNA-Seq performed in a variety of growth conditions. RESULTS: The pRNA-Seq library revealed known tRNA, rRNA and transfer-messenger RNA (tmRNA) processing sites, together with previously uncharacterized RNA cleavage events that were found disproportionately near the 5' ends of transcripts associated with basic bacterial functions such as oxidative phosphorylation and purine metabolism. The majority (97%) of the processed mRNAs were cleaved at precise codon positions within defined sequence motifs indicative of distinct endonucleolytic activities. The most abundant of these motifs corresponded closely to an E. coli RNase E site previously established in vitro. Using the dRNA-Seq library, we performed an operon analysis and predicted 3159 potential TSS. A correlation analysis uncovered 105 antiparallel pairs of TSS that were separated by 18 bp from each other and were centered on single palindromic TAT(A/T)ATA motifs (likely - 10 promoter elements), suggesting that, consistent with previous in vitro experimentation, these sites can initiate transcription bi-directionally and may thus provide a novel form of transcriptional regulation. TSS and RNA-Seq analysis allowed us to confirm expression of small non-coding RNAs (ncRNAs), many of which are differentially expressed in swarming and biofilm formation conditions. CONCLUSIONS: This study uses pRNA-Seq, a method that provides a genome-wide survey of RNA processing, to study the bacterium Pseudomonas aeruginosa and discover extensive transcript processing not previously appreciated. We have also gained novel insight into RNA maturation and turnover as well as a potential novel form of transcription regulation. NOTE: All sequence data has been submitted to the NCBI sequence read archive. Accession numbers are as follows: [NCBI sequence read archive: SRX156386, SRX157659, SRX157660, SRX157661, SRX157683 and SRX158075]. The sequence data is viewable using Jbrowse on www.pseudomonas.com .

Fluorogenic RNA Mango aptamers for imaging small non-coding RNAs in mammalian cells.

Despite having many key roles in cellular biology, directly imaging biologically important RNAs has been hindered by a lack of fluorescent tools equivalent to the fluorescent proteins available to study cellular proteins. Ideal RNA labelling systems must preserve biological function, have photophysical properties similar to existing fluorescent proteins, and be compatible with established live and fixed cell protein labelling strategies. Here, we report a microfluidics-based selection of three new high-affinity RNA Mango fluorogenic aptamers. Two of these are as bright or brighter than enhanced GFP when bound to TO1-Biotin. Furthermore, we show that the new Mangos can accurately image the subcellular localization of three small non-coding RNAs (5S, U6, and a box C/D scaRNA) in fixed and live mammalian cells. These new aptamers have many potential applications to study RNA function and dynamics both in vitro and in mammalian cells.

Purification of RNA Mango Tagged Native RNA-protein Complexes from Cellular Extracts Using TO1-Desthiobiotin Fluorophore Ligand.

A native purification strategy using RNA Mango for RNA based purification of RNA-protein complexes is described. The RNA Mango aptamer is first genetically engineered into the RNA of interest. RNA Mango containing complexes obtained from cleared cellular native extracts are then immobilized onto TO1-Desthiobiotin saturated streptavidin agarose beads. The beads are washed to remove non-specific complexes and then the RNA Mango containing complexes are eluted by the addition of free biotin to the beads. Since the eluted complexes are native and fluorescent, a second purification step such as size exclusion chromatography can easily be added and the purified complexes tracked by monitoring fluorescence. The high purity native complexes resulting from this two-step purification strategy can be then used for further biochemical characterization.

Transitioning to DNA genomes in an RNA world.

The unexpected ability of an RNA polymerase ribozyme to copy RNA into DNA has ramifications for understanding how DNA genomes evolved.

Ribonucleoprotein purification and characterization using RNA Mango.

The characterization of RNA-protein complexes (RNPs) is a difficult but increasingly important problem in modern biology. By combining the compact RNA Mango aptamer with a fluorogenic thiazole orange desthiobiotin (TO1-Dtb or TO3-Dtb) ligand, we have created an RNA tagging system that simplifies the purification and subsequent characterization of endogenous RNPs. Mango-tagged RNP complexes can be immobilized on a streptavidin solid support and recovered in their native state by the addition of free biotin. Furthermore, Mango-based RNP purification can be adapted to different scales of RNP isolation ranging from pull-down assays to the isolation of large amounts of biochemically defined cellular RNPs. We have incorporated the Mango aptamer into the S. cerevisiae U1 small nuclear RNA (snRNA), shown that the Mango-snRNA is functional in cells, and used the aptamer to pull down a U1 snRNA-associated protein. To demonstrate large-scale isolation of RNPs, we purified and characterized bacterial RNA polymerase holoenzyme (HE) in complex with a Mango-containing 6S RNA. We were able to use the combination of a red-shifted TO3-Dtb ligand and eGFP-tagged HE to follow the binding and release of the 6S RNA by two-color native gel analysis as well as by single-molecule fluorescence cross-correlation spectroscopy. Together these experiments demonstrate how the Mango aptamer in conjunction with simple derivatives of its flurophore ligands enables the purification and characterization of endogenous cellular RNPs in vitro.

Structural basis for high-affinity fluorophore binding and activation by RNA Mango.

Genetically encoded fluorescent protein tags have revolutionized proteome studies, whereas the lack of intrinsically fluorescent RNAs has hindered transcriptome exploration. Among several RNA-fluorophore complexes that potentially address this problem, RNA Mango has an exceptionally high affinity for its thiazole orange (TO)-derived fluorophore, TO1-Biotin (Kd ∼3 nM), and, in complex with related ligands, it is one of the most redshifted fluorescent macromolecular tags known. To elucidate how this small aptamer exhibits such properties, which make it well suited for studying low-copy cellular RNAs, we determined its 1.7-Å-resolution co-crystal structure. Unexpectedly, the entire ligand, including TO, biotin and the linker connecting them, abuts one of the near-planar faces of the three-tiered G-quadruplex. The two heterocycles of TO are held in place by two loop adenines and form a 45° angle with respect to each other. Minimizing this angle would increase quantum yield and further improve this tool for in vivo RNA visualization.